KR20240017854A - System and method for sequencing nucleotides using two optical channels - Google Patents

Abstract

The disclosed technology relates to the field of nucleic acid sequencing, and more particularly to systems and methods for DNA sequencing utilizing single optical excitation and at least three fluorescent labels. In some embodiments, the disclosed technology includes a first nucleotide coupled to a first fluorescent label capable of emitting light detectable by a first detector, a second fluorescent label capable of emitting light detectable by a second detector. a second nucleotide coupled to a third nucleotide coupled to a third fluorescent label capable of emitting light detectable by both the first and second detectors, and a fourth nucleotide not coupled to the fluorescent label. do. The disclosed technology can identify nucleotides in a nucleic acid sequence based on whether the emission is received by the first detector, the second detector, both the first and second detectors, or neither the first nor the second detector. You can.

Description

System and method for sequencing nucleotides using two optical channels

Cross-references with related applications

This application claims priority to U.S. Patent Application No. 17/338,590, filed June 3, 2021, the contents of which are incorporated by reference in their entirety.

In some types of next-generation sequencing technologies, DNA clusters are generated on a flowcell after amplification of target polynucleotides. Increasing the density of DNA clusters within the flowcell and deploying faster imaging technologies (e.g., through the use of nanowells) can scale DNA sequencing throughput and reduce overall sequencing costs. However, with faster imaging techniques, the signal from DNA clusters may become dimmer, requiring a higher power light source to compensate for the darker signal. High-power light sources, such as high-power lasers, are expensive, consume relatively large amounts of energy, and can generate significant amounts of heat that must be dissipated. Additionally, exposure to higher output light may cause more light-induced damage to the target polynucleotide, resulting in faster signal decay and reduced sequencing data quality over multiple sequencing cycles.

Existing DNA sequencing systems and methods, for example, existing sequencing platforms using two- or four-channel sequencing chemistries, utilize two or more excitation light sources to excite deoxyribonucleic acid analogs conjugated with a fluorescent label at a target polynucleotide. You can. Reducing the number of excitation light sources can reduce cost and increase the performance robustness of such sequencing systems. Additionally, reducing the number of excitation light sources can reduce unnecessary exposure of the sample to light, thereby reducing DNA damage caused by light. In one aspect, a system for identifying nucleotides in a polynucleotide bound to a substrate and methods of using such system are disclosed. The system includes a first detector configured to detect light in a first wavelength range; a second detector configured to detect light in a second wavelength range; A light source including a laser or light-emitting diode that outputs light at an optical frequency; and a processor. The processor generates light at optical frequencies to stimulate emission from the nucleic acid sequences on the substrate; It can be configured to identify nucleotides in a nucleic acid sequence based on whether the emission is received by a first detector, a second detector, both the first and second detectors, or neither the first nor the second detector. The system includes a first nucleotide coupled to a first fluorescent label; a second nucleotide coupled to a second fluorescent label; a third nucleotide coupled to a third fluorescent label; And it may further include a fourth nucleotide that is not coupled to the fluorescent label. The light source excites the first fluorescent label to emit light that can be detected by the first detector; exciting the second fluorescent label to emit light that can be detected by a second detector; The third fluorescent label may be configured to excite to emit light detectable by both the first and second detectors.

Another embodiment includes emitting light at an optical frequency from a light source to a polynucleotide; determining whether the polynucleotide has a bound fluorescent label that emits fluorescence under a first wavelength of light, a second wavelength of light, both the first and second wavelengths of light, or is devoid of fluorescence; Determining the sequence of a polynucleotide comprising identifying the sequence of the polynucleotide based on whether there is detectable emission or no fluorescence in the first wavelength of light, the second wavelength of light, both the first and second wavelengths of light. This is how to do it.

The systems, devices, kits and methods disclosed herein each have multiple aspects, no single one of which is solely responsible for the desirable properties. Numerous other embodiments are also contemplated, including embodiments with fewer, additional and/or different components, steps, features, objects, benefits, and advantages. Components, aspects, and steps may be arranged and ordered differently. After considering this discussion, and especially after reading the section titled “Detailed Description,” you will understand how the features of the devices and methods disclosed herein provide advantages over other known devices and methods.

It should be understood that any of the features of the systems disclosed herein may be combined together in any desirable manner and/or configuration. Additionally, it should be understood that any of the features of the methods disclosed herein may be combined together in any desirable manner. Additionally, it should be understood that any combination of features of the methods and/or systems may be used together and/or combined with any of the embodiments disclosed herein. It should be understood that all combinations of the foregoing concepts and additional concepts discussed in more detail below are considered part of the inventive subject matter disclosed herein and may be used to achieve the benefits and advantages described herein.

Features of embodiments of the present disclosure will become apparent by reference to the following detailed description and drawings, in which like reference numerals correspond to similar, but not identical, elements. For the sake of brevity, reference numerals or features having functions described above may or may not be described in relation to the other drawings in which they appear.
1A schematically depicts an example sequencing system capable of performing embodiments of the disclosed sequencing technology.
1B schematically depicts an example imaging system that may be used in embodiments of the disclosed sequencing technology.
1C schematically depicts another example imaging system that may be used with embodiments of the disclosed sequencing technology.
Figure 2 shows a functional block diagram of an example computer system used in a sequencing system as shown in Figure 1A.
Figure 3 depicts an exemplary dye labeling scheme for an embodiment of the disclosed sequencing technology.
Figure 4 shows an exemplary emission spectrum of a fully functionalized set of nucleotides within an embodiment of the disclosed sequencing technology.
Figure 5 schematically shows an example of the fluorescence results of a single excitation, two optical channel detection of three fully functionalized nucleotides.
Figure 6 shows scatter plot results of a sequencing experiment performed according to one embodiment of the disclosed technology.
7A and 7B are line graphs showing the results of a sequencing experiment performed according to one embodiment of the disclosed technology.
Figure 8 shows scatter plot results of additional sequencing experiments performed according to one embodiment of the disclosed technology.
Figure 9A shows scatter plot results of an alternative additional sequencing experiment performed in accordance with an embodiment of the disclosed technology.
9B and 9C are line graphs showing the results of alternative additional sequencing experiments conducted in accordance with embodiments of the disclosed technology.

All patents, patent applications, and other publications mentioned herein, including all sequences disclosed in such references, are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually incorporated by reference. is explicitly included by reference. All documents cited are, in relevant part, incorporated herein by reference in their entirety for the purposes specified in the context in which they are cited herein. However, citation of any document should not be construed as an admission that the document is prior art with respect to this disclosure.

introduction

Embodiments of the disclosed technology relate to next-generation sequencing systems and methods capable of identifying four nucleotide bases using a single excitation light source and two different optical channels. The disclosed sequencing technology may use a sequencing process by synthesis. During each sequencing cycle, four types of nucleotide analogs can be incorporated into the growth primers that hybridize to the polynucleotide being sequenced. In some embodiments, the four types of nucleotide analogs include a first deoxyguanosine triphosphate (dGTP) analog that is not conjugated to any fluorescent dye, a first deoxythymidine triphosphate (dTTP) analog that is conjugated to a first fluorescent dye, and a first fluorescent dye. 2 a deoxytidine triphosphate (dCTP) analog conjugated with a fluorescent dye, and a third deoxyadenosine triphosphate (dATP) analog conjugated with a fluorescent dye. The fluorescent dyes conjugated to the four types of nucleotide analogs are illustrative only and are not intended to be limiting. In other embodiments, the nucleotide analog that is not conjugated to any fluorescent dye may be dTTP, dCTP, or dATP. In other embodiments, the nucleotide analog conjugated to the first fluorescent dye may be dGTP, dCTP, or dATP. In other embodiments, the nucleotide analog conjugated with the second fluorescent dye may be dGTP, dTTP, or dATP. In other embodiments, the nucleotide analog conjugated with the third fluorescent dye can be dGTP, dTTP, or dCTP.

The three fluorescent dyes can be excited by a single wavelength (or a single narrow wavelength band) of excitation light from a light source such as a laser. The first fluorescent dye has an emission spectrum that can be captured in a first image taken in the first optical channel. The second fluorescent dye has an emission spectrum that can be captured in a second image taken in the second optical channel. The third fluorescent dye has an emission spectrum that is wide enough to be captured in images captured in both the first and second optical channels. Accordingly, a nucleotide analog (or a DNA cluster having a plurality of identical nucleotide analogs) associated with a free dye, a first dye, a second dye, or a third dye may be image-free, the first image, the second image, or both images, respectively. It can be identified depending on whether a diffraction limit point occurs.

Non-limiting advantages of the disclosed systems and methods include enabling a more efficient sequencing workflow with fewer processing steps. Additionally, sequencing systems using a three-dye system as described herein may have fewer components, be less expensive to operate, and be more power efficient. For example, the disclosed systems and methods may require fewer excitation light sources than previous systems. In some embodiments, only a single excitation light source may be required compared to previous systems that required multiple excitation light sources. This reduces the number of imaging steps required and allows the system to be more power efficient. Having fewer components in a sequencer can substantially reduce costs and simplify device design. Fewer components in the sequencer can increase system efficiency and robustness of device performance. Additionally, the disclosed systems and methods may require less exposure of the target polynucleotide to excitation light, which may reduce light-induced DNA damage and thereby increase sequencing data quality and sequence base calling accuracy.

Example sequencer

1A, an exemplary sequencing system 100 capable of performing the disclosed sequencing techniques is shown. Sequencing system 100 may be configured to utilize the disclosed sequencing method based on a single optical excitation and at least three fluorescent labels. Non-limiting examples of sequencing reactions utilized may include variations of sequencing processes by synthesis, such as those used for Illumina® dye sequencing or HeliScope® single molecule sequencing.

Sequencing system 100 may include an optical system 102 configured to generate raw sequencing data using sequencing reagents supplied by a fluidics system 104 that is part of sequencing system 100. Raw sequencing data may include fluorescence images captured by optical system 102. Sequencing system 100 may further include a computer system 106 that may be configured to control optical system 102 and fluidics system 104 via communication channels 108a and 108b. For example, computer interface 110 of optical system 102 may be configured to communicate with computer system 106 via communication channel 108a.

During a sequencing reaction, the fluidics system 104 may direct the flow of reagents through one or more reagent tubes 112 to or from a flow cell 114 disposed on the loading stage 116. Reagents may include, for example, fluorescently labeled nucleotides, buffers, enzymes, and cleavage reagents. Flow cell 114 may include at least one fluid channel. Flow cell 114 may be a patterned array flow cell or a random array flow cell. Flow cell 114 may include a plurality of clusters of single-stranded polynucleotides to be sequenced in at least one fluidic channel. The length of the polynucleotide may vary, for example, from 200 bases to 1000 nucleotides. Polynucleotides may be attached to one or more fluidic channels of flow cell 114. In some embodiments, flowcell 114 may include a plurality of wells, where each well may contain multiple copies of the polynucleotide to be sequenced. Mounting stage 116 may be configured to allow proper alignment and movement of flow cell 114 relative to other components of optical system 102. In one embodiment, mounting stage 116 can be used to align flow cell 114 with lens 118.

Optical system 102 may include a single light source 120, such as a single laser or a single LED, configured to produce light with a narrow distribution of wavelengths around a predetermined wavelength, for example, 455 nm. In some embodiments, the predetermined wavelength is in the range of 405 nm to 460 nm. However, embodiments are not limited to any specific wavelength of light. The light source simply needs to be configured to produce light of the correct wavelength to excite the fluorescent label attached to the nucleotides in the flow cell.

Light generated from the light source 120 may pass through the fiber optic cable 122 to excite the fluorescent label in the flow cell 114. The lens 118 mounted on the focuser 124 can move along the z-axis. The focused fluorescence emission can be detected by a detector 126, for example a charge coupled device (CCD) sensor or a complementary metal oxide semiconductor (CMOS) sensor. In some embodiments, nucleotide incorporation is described in, e.g., Levene et al. Science 299, 682-686 (2003); Lundquist et al. Opt. Lett. 33, 1026-1028 (2008); and Korlach et al. Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), which disclosure is incorporated herein by reference in its entirety.

Filter assembly 128 of optical system 102 may be configured to filter fluorescent emission from a fluorescent label within flow cell 114. Filter assembly 128 may include a plurality of optical filters that can be selected by the user depending on the specific fluorophore used in the sequencing reaction. In one other embodiment, computer system 106 is configured, for example, by scanning labels and/or barcodes affixed to sample vials and determining specific fluorophores to be used in the sequencing reaction based on the labels and/or barcodes, or by previously By retrieving information stored in memory related to the sequencing reaction, the optical filter to be used in the sequencing reaction can be automatically determined and the filter assembly 128 can then be controlled to select and use the desired optical filter. You can use more than one filter at a time. Each filter may be a long-pass filter, a short-pass filter, a band-stop filter, or a band-pass filter depending on the type of fluorescent molecule used in the system. For example, the user can select a first filter and a second filter. The first filter may be a band-pass filter selected to match the emission spectrum peak of the first fluorescent label. The second filter may be a band-pass filter selected to match the peak of the emission spectrum of the second fluorescent label. The spacing between the transmission windows of the two bandpass filters is, for example, at least 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 nm, or these values. It can be a number or range between any two values. The center of the transmission window of the first band-pass filter and the center of the transmission window of the second band-pass filter may be separated from each other, for example, in the range of 10 nm to 100 nm. The center of the transmission window of the first band-pass filter and the center of the transmission window of the second band-pass filter are 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 nm, or a number or range between any two of these values, or approximately these values. can be separated from

In some embodiments, detector 126 includes one sub-detector, and the filters of filter assembly 128 can be mechanically switched or rotated in front of the sub-detector, such that differently filtered images can be sequentially taken by the sub-detectors. . In some embodiments, detector 126 may include one sub-detector and filter assembly 128 may include at least one layer of a switchable material having variable optical transmittance depending on the application of a stimulus, where the stimulus is light, electrical, , temperature, or any combination thereof. As a result, the filter assembly 128 can provide a plurality of optical filters so that differently filtered images can be sequentially captured by the sub-detector. In some embodiments, detector 126 may include one sub-detector and filter assembly 128 may include one or more switchable filters based on microelectromechanical system technology, such that differently filtered images may be sequentially detected by the sub-detectors. can be filmed.

In some embodiments, detector 126 may include two or more sub-detectors, e.g., a first detector coupled with a first filter and a second detector coupled with a second filter, and the optical system 102 is configured to emit fluorescence. It may include two or more two-color mirrors/beam splitters configured to split. After splitting the fluorescence emission with a two-color mirror, detector 126 can take two differently filtered images simultaneously (or close in time), for example using two sub-detectors coupled with two different filters. You can. In some embodiments, detector 126 may include two or more sub-detectors stacked along the incoming direction of fluorescence emission. Different wavelengths of fluorescence emission may be differentially attenuated or differentially absorbed along the inflow direction, so sub-detectors at different locations along the inflow direction may produce differently filtered images simultaneously (or close in time). It may be configured to take pictures.

In use, a sample with polynucleotides to be sequenced may be loaded into the flow cell 114 and placed on a mounting stage 116. Computer system 106 may then initiate fluidics system 104 to begin the sequencing cycle. During a sequencing reaction, computer system 106 may instruct fluidics system 104 via communication interface 108b to supply reagents, such as labeled nucleotide analogs, to flow cell 114. Via communication interface 108a and computer interface 110, computer system 106 controls light source 120 of optical system 102 to generate light near a predetermined wavelength, e.g., nucleotides incorporated into growth primers hybridized to polynucleotides being sequenced. Analogs can be excited. Computer system 106 may control detector 126 of optical system 102 to capture images of diffraction limited spots of DNA clusters with fluorescently labeled nucleotide analogs. Computer system 106 may receive fluorescence images from detector 126 and process the received fluorescence images to determine the nucleotide sequence of the polynucleotide being sequenced.

Figure 1B shows an example of an imaging system 10000 to be used in the disclosed sequencing technology. For example, imaging system 10000 may be used in the example sequencing system 100 shown in FIG. 1A. Imaging system 10000 may include a light source 11000 capable of providing light to excite fluorophores at a target point on the sample. So that the light source 11000 can provide light of various wavelengths, the light source 11000 may include one or more lasers, light emitting diodes, or other light sources. In some embodiments, light source 11000 may be configured to selectively provide light having a predetermined wavelength range tuned to the set of fluorophores used. In some embodiments, light source 11000 may be configured to output light at an optical frequency that corresponds to a predefined range of wavelengths of light. In some embodiments, a user of the disclosed sequencing system can select a specific optical frequency to be output from light source 11000 depending on the specific fluorophore used in the sequencing reaction.

Imaging system 10000 may include a microfluidic device including an optical path 12000 from a light source 11000 to a sample 13000, for example, one or more flow chambers in which one or more sequencing reactions occur. In some embodiments, the optical path 12000 can be configured to direct light from a mirror, lens, prism, quarter wave plate, half wave plate, polarizer, filter, dichroic mirror, beam splitter, beam combiner, objective lens, light source relative to a sample, etc. The light path 12000 may be configured to direct light from the light source 11000 to the sample 13000. Additionally, optical path 12000 may include optical components that may be configured to direct light emitted from sample 13000 to integrated detection system 15000. In some embodiments, some of the optical elements used to direct light from light source 11000 to sample 13000 are also used to direct light from sample 13000 to integrated detection system 15000. Additional examples of optical paths and optical systems can be found in U.S. Patent No. 7,589,315, U.S. Patent No. 8,951,781, or U.S. Patent No. 9,193,996, each of which is incorporated herein by reference in its entirety.

The imaging system 10000 may include a scanning system 14000 that effectively moves light relative to the sample 13000 to scan the sample to generate an image. In some embodiments, scanning system 14000 may be implemented within optical path 12000. For example, scanning system 14000 may include one or more scanning mirrors that move relative to each other within optical path 12000 to effectively move light from light source 11000 across the sample. In some embodiments, scanning system 14000 may be implemented as a mechanical system that physically moves sample 13000 such that the sample moves relative to light from light source 11000. In some embodiments, the scanning system 14000 may be a combination of optical components in the light path 12000 and a mechanical system for physically moving the sample 13000 such that light from the light source 11000 and the sample 13000 move relative to each other.

Imaging system 10000 may include one or more photodetectors, as well as an integrated detection system 15000 that includes associated electronic circuitry, a processor, data storage, memory, etc. to acquire and process image data of the sample 13000. In some embodiments, integrated detection system 15000 may include a photomultiplier tube, an avalanche photodiode, an image sensor (e.g., CCD, CMOS sensor, etc.). In some embodiments, the optical detector of the integrated detection system 15000 may include components that amplify optical signals and may be sensitive to single photons. In some embodiments, the photo detector of integrated detection system 15000 may have multiple channels or pixels. Integrated detection system 15000 may acquire one or more images based on detected light from sample 13000.

In some embodiments, optical path 12000 can include an array generator 12100 that can generate a plurality of exposure areas on sample 13000. In some embodiments, array generator 12100 can generate a specific light exposure pattern on sample 13000. Scanning system 14000 may be used to scan the exposed area over sample 13000 to selectively illuminate areas of sample 13000 for imaging. Integrated detection system 15000 may integrate signals corresponding to specific points on sample 13000 as multiple exposure areas are scanned over sample 13000. For example, for individual points in sample 13000, integral detection system 1500 may selectively aggregate detection signals corresponding to individual points where the individual points are illuminated at different times by different exposure areas. In some embodiments, the combination of array generator 12100 and integrated detection system 15000 can detect light from multiple points in sample 13000 simultaneously or near simultaneously. In some embodiments, the combination of array generator 12100 and integrated detection system 15000 may integrate light detected at multiple points in the sample over time.

In some embodiments, multiple sequencing reactions may be run in parallel in multiple flow chambers of Sample 13000. For example, multiple sequencing reactions can be performed on multiple biological samples. In some embodiments, multiple sequencing reactions may use different sets of fluorophores. In some embodiments, the light source 11000, the array generator 12100, and the scanning system 14000 are configured to illuminate different regions of the sample 13000 with light of different optical frequencies depending on the different sets of fluorophores used in the sequencing reactions occurring in different regions of the sample 13000. It can be configured to selectively illuminate an area.

Figure 1C shows another example of an imaging system 1500 to be used in the disclosed sequencing technology. For example, imaging system 1500 may be used in the example sequencing system 100 shown in FIG. 1A. Imaging system 1500 can be used to image a flow cell 1600 having an upper layer 1671 and a lower layer 1673 that can be separated by a fluid-filled channel 1675. In the depicted configuration, the upper layer 1671 may be optically transparent and light from the imaging system 1500 may be focused on an area 1676 on the inner surface 1672 of the upper layer 1671. In an alternative configuration, light from the imaging system 1500 may be focused on the inner surface 1674 of the lower layer 1673. One or both surfaces may include sequence features containing polynucleotides and sequencing reactions that can be detected by imaging system 1500.

Imaging system 1500 may include an objective lens 1501 configured to direct excitation light from light source 1502 to flow cell 1600 and emission from flow cell 1600 to detector 1508. In the example layout, excitation light from light source 1502 passes through lens 1505, then through beam splitter 1506, then through objective 1501 and into flow cell 1600. In some embodiments, light source 1502 may include one or more lasers, light emitting diodes, or any combination thereof. For example, light source 1502 may include one laser 1503 and one light emitting diode 1504, which may provide light at different wavelengths or ranges of wavelengths that a user can select. Light emitted from flow cell 1600 may be captured by objective lens 1501 and reflected by a beam splitter through beam conditioning optics 1507 and reflected to detector 1508 (e.g., CMOS sensor). Beam splitter 1506 may direct the emission light in a direction orthogonal to the path of the excitation light. The position of the objective lens 1501 can be moved in the z dimension to change the focus of the excitation light of the flow cell 1600. The imaging system 1500 can capture images of multiple areas of the flow cell 1600 by moving back and forth in the y direction.

The computer system 106 of the example sequencing system 100 shown in FIG. 1A may be configured to control the optical system 102 and the fluidic system 104. Although many configurations are possible for computer system 106, one embodiment is illustrated in FIG. 2. As shown in FIG. 2 , computer system 106 may include a processor 202 in electrical communication with memory 204, storage 206, and a communication interface 208.

Processor 202 may be configured to execute instructions that cause fluidics system 104 to supply reagents to flow cell 114 during a sequencing reaction. The processor 202 may control the light source 120 of the optical system 102 to execute instructions to generate light near a predetermined wavelength. Processor 202 may execute instructions to control detector 126 of optical system 102 and receive data from detector 126. Processor 202 may execute instructions to process data received from detector 126, such as a fluorescence image, and determine the nucleotide sequence of a polynucleotide based on the data received from detector 126.

Memory 204 may be configured to store instructions for configuring processor 202 to perform the functions of computer system 106 when sequencing system 100 is powered on. When sequencing system 100 is powered off, storage 206 may store instructions for configuring processor 202 to perform the functions of computer system 106. Communication interface 208 may be configured to facilitate communication between computer system 106, optical system 102, and fluidics system 104.

The computer system 106 may include a user interface 210 configured to communicate with a display device (not shown) for displaying sequencing results of the single sequencing system 100. User interface 210 may be configured to receive input from a user of sequencing system 100. Optical system interface 212 and fluidics system interface 214 of computer system 106 may be configured to control optical system 102 and fluidics system 104 via communication links 108a and 108b shown in FIG. 1A. For example, optical system interface 212 may communicate with computer interface 110 of optical system 102 via communication link 108a.

Computer system 106 may include a nucleobase determiner 216 configured to determine the nucleotide sequence of the polynucleotide using data received from detector 126. The nucleobase determiner 216 may include one or more of a template generator 218, a position registerer 220, an intensity extractor 222, an intensity corrector 224, a base interrogator 226, and a quality score determiner 228. Template generator 218 may be configured to generate a template for the location of polynucleotide clusters on flow cell 114 using the fluorescence image captured by detector 126. Position registerer 220 may be configured to register the positions of polynucleotide clusters in flow cell 114 to the fluorescence image captured by detector 126 based on the position template generated by template generator 218. Intensity extractor 222 may be configured to extract the intensity of fluorescence emission from the fluorescence image to generate the extracted intensity. Intensity corrector 224 may be configured to reduce or eliminate cross-talk between fluorescent labels in different optical channels, for example, by color correcting the extracted intensities to produce corrected intensities. In some embodiments, intensity corrector 224 may phase correct or pre-phase correct the extracted intensity. Base interrogator 226 may be configured to determine the nucleobase of the polynucleotide from the modified intensity. The bases of the polynucleotide determined by base caller 226 may be associated with a quality score determined by quality score determiner 228. Additional details of calculations that can be performed by nucleobase determiners can be found in U.S. Patent Application Publication Nos. 2020/0080142 and 2012/0020537, each of which is incorporated herein by reference in its entirety.

Sequencing using single excitation, 3-label, 2-optical channels

The disclosed technology may use a sequencing-by-synthesis process. During each sequencing cycle, four types of nucleotide analogs can be added and incorporated into the growing primer-polynucleotide. The four types of nucleotide analogs can have different modifications. For example, as shown in Figure 3, the first type of nucleotide may be an analog of deoxythymidine triphosphate (dTTP) conjugated to a first type of fluorescent label via a linker. The second type of nucleotide may be an analog of deoxycytidine triphosphate (dCTP) conjugated to a second type of fluorescent label via a linker. The third type of nucleotide may be an analog of deoxyadenosine triphosphate (dATP) conjugated to a third type of fluorescent label via a linker. A fourth type of nucleotide may be an analog of deoxyguanosine triphosphate (dGTP) that is not conjugated with any fluorescent label. After incorporating the nucleotide analogs, the unincorporated nucleotide analogs can be washed and removed.

The first fluorescent label can have an emission spectrum that can be captured in a first image taken in the first optical channel. The second fluorescent label can have an emission spectrum that can be captured in a second image taken in a second optical channel separate from the first optical channel. The third fluorescent dye can have an emission spectrum that can be captured in both the first and second optical channels. In some embodiments, coupling a dye to a nucleotide may not result in significant changes to the absorption or emission spectra. As a result, in the example shown in FIG. 3, it can be confirmed that dATP appears in both the first image and the second image. dGTP may not appear in either image. dTTP can be confirmed to appear only in the first image. dCTP can be confirmed to appear only in the second image.

The exemplary nucleotides shown in Figure 3 may be fully functionalized nucleotides. The linker located between the nucleotide base and the fluorescent molecule may include one or more cleavage groups. The fluorescent label can be removed from the nucleotide analog by cleaving the linker before subsequent sequencing cycles. For example, a linker that attaches a fluorescent label to a nucleotide analog may contain azide and/or alkoxy groups, for example on the same carbon, such that the linker can be cleaved after each cycle of incorporation by the phosphine reagent to release the fluorescent label. You can. Nucleotide triphosphates can be reversibly blocked at the 3' position, so sequencing is controlled, and only a single nucleotide analog can be added to each extension primer-polynucleotide in each cycle. For example, the 3' ribose position of a nucleotide analog may contain both alkoxy and azido functional groups that can be removed by cleavage with a phosphine reagent to generate a nucleotide that can be further expanded. Before subsequent sequencing cycles, another nucleotide analog can be added to each extension primer-polynucleotide by removing the reversible 3' block.

Figure 4 shows an exemplary emission spectrum of a set of fully functionalized nucleotides that can be used in embodiments of a single excitation, three-label, two-optical channel sequencing method. In this example, fully functionalized dTTP (ffT) is labeled with a first dye whose emission spectrum is plotted as a curve with a peak at approximately 500 nm when excited by a 450 nm light source. Fully functionalized dATP (ffA) is labeled with a second dye whose emission spectrum is displayed as a curve with a peak at approximately 575 nm when excited by a 450 nm light source. In this example, dGTP is not labeled with any fluorescent dye. No. Fully functionalized dCTP (ffC) is labeled with a third dye with a relatively broad emission spectrum shown as a curve with a broad peak at approximately 535 nm when excited by a 450 nm light source. In some embodiments, the emission spectrum of the third dye emits photons over a wider range of wavelengths compared to the emission spectrum of the first or second dye. In an alternative embodiment, a third dye with a broader emission spectrum is used in a two-excitation, two-optical channel sequencing method, e.g., a laser producing light in the blue range and a laser producing light in the green range are both used. It can be used in a method to excite dyes.

In Figure 4, the first optical channel is represented by a window spanning from about 450 nm to about 530 nm. The second optical channel is represented by a window spanning from about 545 nm to about 650 nm. Therefore, when excited by a 450 nm light source, ffT emission generates a peak signal (e.g., power) in the first optical channel and only a small or negligible signal in the second optical channel. The ffA emission produces a peak signal in the second optical channel and only a small or negligible signal in the first optical channel. The emission spectrum of the third dye has a wider range compared to the emission spectrum of the first or second dye. Because it has an emission wavelength of , ffC emission generates relatively large signals in both windows corresponding to the first and second optical channels. Because dGTP is not conjugated to any fluorescent label, it does not fluoresce and is not detected in the first or second optical channel. In some embodiments, the third dye on ffC is selected to be brighter than the other two dyes. As a result, the ffC signal size of the first optical channel will be similar to the ffT signal size of the first optical channel, and the ffC signal size of the second optical channel will be similar to the ffA signal size of the second optical channel.

In some embodiments, one of the three fluorescent dyes may be a regular Stokes shift dye. As used herein, a general Stokes shift dye is a dye having a Stokes shift between 55 and 95 nm or comprising a Stokes shift of about 55, 60, 70, 80, 90, 95 nm or any value in between. refers to One of the three fluorescent dyes may be a short Stokes shift dye. As used herein, short Stokes shift dye refers to a dye that has a Stokes shift of about 5, 10, 20, 30, 40, 50 nm, or any value in between. One of the three fluorescent dyes may be a long Stokes shift dye. As used herein, long Stokes shift dye refers to a dye with a Stokes shift of about 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm or any value in between. do.

Figure 5 schematically shows an example of single excitation, two-optical channel detection of fully functionalized nucleotides labeled according to the example shown in Figure 4. As shown in Figure 5, a single light source, such as a “blue” laser, can excite three fluorescent labels at a predetermined wavelength, such as 450 nm. In various embodiments, the output optical frequency of a single light source may or may not be tunable. Detection of a fluorescent label may involve capturing the fluorescence emission in two separate optical channels. For example, ffT and ffC are captured in a first image taken in the “blue” channel, represented by a window spanning from about 460 nm to about 530 nm. ffA and ffC are captured in a second image taken in the “green” channel, represented by a window spanning from about 550 nm to about 650 nm. In some embodiments, fluorescence images can be stored for later processing offline. In some embodiments, fluorescence images can be processed in real time to determine the sequence of primer-polynucleotides growing in each cluster.

In some embodiments, the disclosed systems can be used to identify nucleotides in a nucleic acid sequence bound to a substrate. In some embodiments, the disclosed system includes: a first detector configured to detect light in a first wavelength range; a second detector configured to detect light in a second wavelength range; A light source including a laser or light-emitting diode that outputs light at an optical frequency; and a processor. The processor generates light at optical frequencies to stimulate emission from nucleic acid sequences on the substrate; Nucleotides within a nucleic acid sequence are identified based on whether the emission is received by the first detector, the second detector, both the first and second detectors, or neither the first nor the second detector. In some embodiments, the first wavelength range and the second wavelength range do not overlap.

In some embodiments, the optical frequency corresponds to a wavelength of a predefined range of optical wavelengths, where the predefined range includes at least one wavelength that is shorter than all wavelengths in the first range and the second range. In some embodiments, the predefined range includes 405 nm to 460 nm. In some embodiments, the optical frequency corresponds to a wavelength of light in a predefined range of wavelengths, where the predefined range includes at least one wavelength that is longer than some wavelength in the first range or the second range.

In some embodiments, the disclosed systems include a first nucleotide coupled to a first fluorescent label; a second nucleotide coupled to a second fluorescent label; a third nucleotide coupled to a third fluorescent label; And it may further include a fourth nucleotide that is not coupled to the fluorescent label. The light source excites the first fluorescent label to emit light that can be detected by the first detector; exciting the second fluorescent label to emit light that can be detected by a second detector; Exciting the third fluorescent label emits light that can be detected by both the first and second detectors. In some embodiments, the light source is configured to excite the fluorescent label by a two-photon absorption process.

In some embodiments, the processor can be configured to identify nucleotides within a nucleic acid sequence based on emission signal intensities received by the first detector and the second detector. The first fluorescent label can be identified as having a greater signal intensity received by the first detector compared to the signal intensity received by the second detector. The second fluorescent label can be identified by having a greater signal intensity received by the second detector compared to the signal intensity received by the first detector. The third fluorescent label can be identified as having a similar magnitude of signal intensity received by the first detector compared to the signal intensity received by the second detector. The fourth fluorescent label can be identified as having a low signal intensity (e.g., substantially close to background level) received by the first and second detectors.

In some embodiments, the emission spectrum of the third fluorescent label has a wider range of emission wavelengths compared to the emission spectrum of the first or second fluorescent label. In some embodiments, the third fluorescent label is selected to have a greater emission intensity (brighter) than the first or second fluorescent label.

In some embodiments, the first fluorescent label has a Stokes shift between 20 nm and 50 nm, the second fluorescent label has a Stokes shift between 100 nm and 130 nm, and the third fluorescent label has a Stokes shift between 60 nm and 90 nm. has a Stokes shift of . In some embodiments, the first fluorescent label cannot be detected by the second detector, wherein the second fluorescent label cannot be detected by the first detector. In some embodiments, the first fluorescent label can also be detected by the second detector, or the second fluorescent label can also be detected by the first detector.

In some embodiments, the fluorescent label is selected from the group consisting of polymethine derivatives, coumarin derivatives, benzopyran derivatives, chromenoquinoline derivatives, and compounds containing a bis-boron heterocycle, such as BOPPY and BOPYPY. In some embodiments, the fluorescent label is selected from the group consisting of:

, and .

In some embodiments, the disclosed systems may further include an additional first nucleotide that is not coupled to a fluorescent label. The population or concentration of additional first nucleotides not coupled to the fluorescent label is about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% or It can be any value in between. In some embodiments, the disclosed systems can further comprise an additional first nucleotide coupled to an alternative fluorescent label, wherein the alternative fluorescent label can be excited by a light source to emit light detectable by the first detector. does not exist. The population or concentration of additional first nucleotides coupled to the alternative fluorescent label may be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% or It can be any value in between. In some embodiments, the replacement fluorescent label and the first fluorescent label have different fluorescence emission spectra. In some embodiments, the disclosed systems can further comprise an additional first nucleotide coupled to an alternative fluorescent label, wherein the alternative fluorescent label can be excited by a light source to emit light detectable by the first detector. and wherein the alternative fluorescent label emits a dimmer light compared to the first fluorescent label. The population or concentration of additional first nucleotides coupled to the alternative fluorescent label may be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% or It can be any value in between. In some embodiments, the replacement fluorescent label and the first fluorescent label have different fluorescence emission spectra.

In some embodiments, the alternative fluorescent label may be a fluorescent dye that can be excited by a “green” laser, for example a light source with a wavelength between about 490 nm and 550 nm, for example about 532 nm. Non-limiting examples of alternative fluorescent labels are disclosed in U.S. Pat. No. 10,982,261, which is incorporated by reference in its entirety. In one example, the alternative fluorescent label has the following structure: . Other alternative fluorescent labels include those that can be excited by a “red” laser, for example a light source with a wavelength of about 630 nm to about 700 nm, for example about 660 nm.

In some embodiments, the disclosed systems may further include an additional second nucleotide that is not coupled to a fluorescent label. The population or concentration of additional second nucleotides that are not coupled to the fluorescent label may be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% or It can be any value in between. In some embodiments, the disclosed systems can further comprise an additional second nucleotide coupled to an alternative fluorescent label, wherein the alternative fluorescent label can be excited by a light source to emit light detectable by a second detector. does not exist. The population or concentration of the additional second nucleotides coupled to the alternative fluorescent label may be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% or It can be any value in between. In some embodiments, the replacement fluorescent label and the second fluorescent label have different fluorescence emission spectra. In some embodiments, the disclosed systems can further comprise an additional second nucleotide coupled to an alternative fluorescent label, wherein the alternative fluorescent label can be excited by a light source to emit light detectable by a second detector. and the alternative fluorescent label emits darker light compared to the second fluorescent label. The population or concentration of the additional second nucleotides coupled to the alternative fluorescent label may be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% or It can be any value in between. In some embodiments, the replacement fluorescent label and the second fluorescent label have different fluorescence emission spectra.

In some embodiments, the disclosed systems may further include an additional third nucleotide that is not coupled to a fluorescent label. The population or concentration of additional third nucleotides that are not coupled to the fluorescent label may be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% of the total population or concentration of third nucleotides. It can be any value in between. In some embodiments, the disclosed systems may further comprise an additional third nucleotide coupled to an alternative fluorescent label, wherein the alternative fluorescent label is excited by a light source to emit light detectable by the first or second detector. cannot be released The population or concentration of additional third nucleotides coupled to the alternative fluorescent label may be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% or It can be any value in between. In some embodiments, the alternative fluorescent label and the third fluorescent label have different fluorescence emission spectra. In some embodiments, the disclosed systems may further comprise an additional third nucleotide coupled to an alternative fluorescent label, wherein the alternative fluorescent label is excited by a light source to emit light detectable by the first and second detectors. and wherein the alternative fluorescent label emits a dimmer light compared to the third fluorescent label. The population or concentration of additional third nucleotides coupled to the alternative fluorescent label may be about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50% or It can be any value in between. In some embodiments, the alternative fluorescent label and the third fluorescent label have different fluorescence emission spectra.

In some embodiments, the detector may include a complementary metal-oxide-semiconductor image sensor, a charge coupled device image sensor, a photomultiplier tube, a photodiode, or any combination thereof. In some embodiments, the disclosed systems may further include one or more optical filter materials, one or more diffraction gratings, one or more light dispersing elements, or any combination thereof. In some embodiments, the disclosed systems may further comprise a polymerase configured to replicate or transcribe a portion of a nucleic acid sequence by incorporating nucleotides. In some embodiments, the substrate includes a plurality of chemically functionalized regions, a plurality of cavities, a plurality of optical resonators, a plurality of optical waveguides, or any combination thereof. In some embodiments, the fluorescent label is attached to the nucleotide via a cleavable linker. In some further embodiments, the labeled nucleotide may optionally have a fluorescent label attached to the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base via a cleavable linker moiety. For example, the nucleobase may be 7-deaza adenine and the dye is optionally attached to 7-deaza adenine at the C7 position via a cleavable linker. The nucleobase may be 7-deaza guanine and the dye is optionally attached to 7-deaza guanine at the C7 position via a cleavable linker. The nucleobase may be cytosine and the dye is optionally attached to cytosine at the C5 position via a cleavable linker. As another example, the nucleobase can be thymine or uracil and the dye is optionally attached to thymine or uracil at the C5 position via a cleavable linker. In some further embodiments, the cleavable linker has a similar or identical chemical moiety as the reversible terminator 3' hydroxy blocker such that the 3' hydroxy blocker and the cleavable linker can be removed under the same reaction conditions or with a single chemical entity. It can be included. Non-limiting examples of cleavable linkers include the LN3 linker, sPA linker, and AOL linker, each of which is illustrated below.

,

,

In some embodiments, the nucleotide is selected from the group consisting of dGTP analogs, dTTP analogs, dUTP analogs, dCTP analogs, and dATP analogs. In some embodiments, the first nucleotide is a first reversibly blocked nucleotide triphosphate (rbNTP), the second nucleotide is a second rbNTP, the third nucleotide is a third rbNTP, and the fourth nucleotide is a fourth rbNTP, Here, the first nucleotide, second nucleotide, third nucleotide, and fourth nucleotide are each different types of nucleotides. In some embodiments, the four rbNTPs are selected from the group consisting of rbATP, rbTTP, rbUTP, rbCTP, and rbGTP. In some embodiments, each of the four rbNTPs includes a modified base and a reversible terminator 3' blocker. Non-limiting examples of 3' blocking groups include azidomethyl (*-CH ₂ N ₃ ), substituted azidomethyl (e.g., *-CH(CHF ₂ )N ₃ or *-CH(CH ₂ F)N ₃ ) and *-CH ₂ -O-CH2-CH=CH ₂ , where the asterisk * indicates a point attachment to the 3' oxygen of the ribose or deoxyribose ring of the nucleotide.

In some embodiments, the disclosed single excitation, three-label, two-optical channel sequencing method can be implemented on an Illumina NextSeq 500®, NextSeq 550®, NextSeq 1000®, NextSeq 2000®, any NovaSeq®, or MiniSeq® system. .

In some embodiments, dTTP is coupled to , ffT has an emission maximum at about 499 nm when excited by light from about 450 nm to about 460 nm.

In some embodiments, dTTP is coupled to, ffT has an emission maximum at about 490 nm when excited by light from about 450 nm to about 460 nm.

In some embodiments, dCTP is coupled to , ffC has an emission maximum at about 540 nm when excited by light from about 450 nm to about 460 nm.

In some embodiments, dATP is coupled to , ffA has an emission maximum at about 580 nm when excited by light from about 450 nm to about 460 nm.

Additional details about dyes and fully functionalized nucleotides can be found in U.S. Patent Application Publication Nos. 2018/0094140 and 2020/0277670, International Patent Application Publication No. 2017/051201, and U.S. Provisional Patent Application Nos. 63/057758 and 63/127061. and the disclosure is incorporated herein by reference in its entirety.

Example

Experiment 1

Figures 6, 7A, and 7B show the results of sequencing experiments performed according to the disclosed techniques consistent with the examples illustrated in Figures 4 and 5. Sequencing runs were performed on an Illumina MiSeq system for approximately 150 sequencing cycles. A 450 nm LED was used as the excitation light source, and the exposure time for each captured image was approximately 1000 ms. In this experiment, dTTP was was coupled to and ffT has an emission maximum at approximately 499 nm, and an additional population of dTTP without dye was used. dCTP is was coupled to, and ffC had an emission maximum at approximately 540 nm. dATP is was bound to, and ffA had an emission maximum at approximately 580 nm. Figure 6 shows a scatter plot of DNA cluster signals extracted from images of a sample flowcell over approximately 150 sequencing cycles. The horizontal axis represents the signal intensity extracted from images taken in the first “blue” optical channel, and the vertical axis represents the signal intensity extracted from images taken in the second “green” optical channel. Clusters that produced a large signal in the first “blue” optical channel and only a small signal in the second “green” optical channel were identified as having a T base because ffT was labeled with the first dye. Clusters that produced a large signal in the second “green” optical channel and only a small signal in the first “blue” optical channel were identified as A bases because ffA was labeled with a second dye. Clusters that produced sufficiently large signals in both the first and second optical channels could be identified as C bases because ffC was labeled with a third dye. Clusters that produced minimal signal in either the first or second optical channel can be identified as having G bases because the dGTP is not labeled with any dye.

Cluster groups as shown in Figure 6 are easily separable, so base calling can be reliably achieved using this single-light source, three-dye sequencing process. Based on the same experimental data shown in Figure 6, Figure 7A shows the DNA cluster signal intensities for clusters identified by T bases and clusters identified by C bases (as shown in the sample flowcell) over the course of the sequencing cycle. represents the average for the identified clusters). As shown, DNA cluster signal intensity decreased with increasing number of sequencing cycles, but signal intensity was still sufficiently high after approximately 150 sequencing cycles. Figure 7b shows the base call error rate (%) as the sequencing cycle progresses. As shown, the error rate increased as the number of sequencing cycles increased, but the error rate remained sufficiently low (less than 2.2%) after approximately 150 sequencing cycles.

Experiment 2

Figure 8 presents a scatterplot similar to that described in relation to Figure 6, but based on the results of additional sequencing experiments. Additional sequencing experiments used conditions similar to those described in conjunction with Figure 6, but the dye used to label dTTP was , ffT has an emission maximum at approximately 490 nm, and an additional population of dye-free dTTP was also used. The relative amount of dTTP with the first dye to dTTP without the dye was approximately 1:3. As shown in Figure 8, using two populations of dTTPs can adjust the shape of the cluster groups in the scatterplot and affect subsequent base calling performance.

Experiment 3

Figure 9A shows a scatterplot similar to that described in relation to Figure 8, but based on the results of an alternative additional sequencing experiment. An alternative additional sequencing experiment used conditions similar to those described in conjunction with Figure 8, but In addition to the dTTP group labeled with a fourth dye, An alternative additional population of dTTP with was used. The fourth dye could not be excited by the 450 nm LED. The relative amount of dTTP with the first dye to dTTP with the fourth dye was approximately 1.25:0.75. As shown in Figure 9A, using two populations of dTTPs can further tune the shape of the cluster groups in the scatterplot and influence subsequent base calling performance. Based on the same experimental data shown in Figure 9A, Figure 9B shows the DNA cluster signal intensities for clusters identified with T bases and clusters identified with C bases (as shown in the sample flowcell) over the course of the sequencing cycle. represents the average for the identified clusters). As shown, DNA cluster signal intensity decreased with increasing number of sequencing cycles, but signal intensity was still sufficiently high after approximately 150 sequencing cycles. Figure 9c shows the base call error rate (%) as the sequencing cycle progresses. As shown, the error rate increased as the number of sequencing cycles increased, but the error rate remained sufficiently low (less than 2.2%) after approximately 150 sequencing cycles.

Sample

In some embodiments, the sample comprises or consists of purified or isolated polynucleotides derived from tissue samples, biological fluid samples, cell samples, etc. Suitable biological fluid samples include blood, plasma, serum, sweat, tears, sputum, urine, phlegm, ear flow, lymph, saliva, cerebrospinal fluid, cerebrospinal fluid, bone marrow suspension, vaginal flow, cervical lavage, and cerebral fluid. , ascites, milk, respiratory secretions, intestinal and urogenital secretions, amniotic fluid, milk, and leukocyte collection samples. In some embodiments, the sample is a sample that can be easily obtained by a non-invasive procedure, such as blood, plasma, serum, sweat, tears, sputum, urine, sputum, otorrhea, saliva, or stool. In certain embodiments, the sample is a peripheral blood sample, or a plasma and/or serum fraction of a peripheral blood sample. In other embodiments, the biological sample is a swab or smear, biopsy specimen, or cell culture. In other embodiments, the sample is a mixture of two or more biological samples, for example, the biological sample may include two or more of a biological fluid sample, a tissue sample, and a cell culture sample. As used herein, the terms “blood,” “plasma,” and “serum” explicitly include fractionated or processed portions thereof. Similarly, when a sample is taken from a biopsy, swab, smear, etc., “sample” expressly includes the processed fraction or portion derived from the biopsy, swab, smear, etc.

In certain embodiments, the sample is a sample from another individual, a sample from a different developmental stage of the same or different individual, a sample from an individual with another disease (e.g., an individual suspected of having cancer or a genetic disorder), a normal samples obtained from individuals, samples obtained at different stages of the individual's disease, samples obtained from individuals who have received different treatments for the disease, samples from individuals exposed to various environmental factors, samples from individuals predisposed to pathology, samples obtained from individuals exposed to infectious agents. It may be obtained from any source, including but not limited to samples.

In one exemplary, but non-limiting, embodiment, the sample is a maternal sample obtained from a pregnant woman, e.g., a pregnant woman. The maternal sample may be a tissue sample, biological fluid sample, or cell sample. In another illustrative but non-limiting embodiment, the maternal sample is a mixture of two or more biological samples, for example, the biological sample may include two or more of a biological fluid sample, a tissue sample, and a cell culture sample.

In certain embodiments, the sample may be obtained from in vivo cultured tissues, cells, or other polynucleotide-containing sources. Cultured samples can be cultures (e.g., tissues or cells) maintained in different media and conditions (e.g., pH, pressure, or temperature), cultures (e.g., tissues or cells) maintained for different periods of time, different factors or reagents ( e.g., a drug candidate or modulator), or cultures of different types of tissues and/or cells.

In some embodiments, use of the disclosed sequencing technology does not include preparation of a sequencing library. In other embodiments, the sequencing techniques contemplated herein involve the preparation of sequencing libraries. In one exemplary approach, sequencing library preparation involves the production of a random set of adapter modified DNA fragments (e.g., polynucleotides) ready to be sequenced.

Sequencing libraries of polynucleotides can be prepared from DNA or RNA, including equivalents or analogs of DNA or cDNA, such as DNA or cDNA, which is complementary DNA or copy DNA generated from an RNA template by the action of reverse transcriptase. The polynucleotide may be from a double-stranded form (e.g., dsDNA, such as a genomic DNA fragment, cDNA, PCR amplification product, etc.), or in certain embodiments, the polynucleotide may be from a single-stranded form (e.g., ssDNA, RNA, etc.). It may have been converted to dsDNA form. By way of example, in certain embodiments single-stranded mRNA molecules can be copied into double-stranded cDNA suitable for use in preparing sequencing libraries. The exact sequence of the primary polynucleotide molecule is generally not critical to the library preparation method and may or may not be known. In one embodiment, the polynucleotide molecule is a DNA molecule. More specifically, in certain embodiments, the polynucleotide molecule represents the entire genetic complement of the organism or substantially the entire genetic complement of the organism and is not a genomic DNA molecule (e.g., cellular DNA, cell-free DNA (cfDNA), etc.). This typically includes both intron and exon sequences (coding sequences) as well as non-coding regulatory sequences such as promoter and enhancer sequences. In certain embodiments, the primary polynucleotide molecule comprises a human genomic DNA molecule, such as a cfDNA molecule present in the peripheral blood of a pregnant subject.

Methods for isolating nucleic acids from biological sources may vary depending on the nature of the source. One skilled in the art can readily isolate nucleic acids from sources as required for the methods described herein. In some cases, it may be advantageous to fragment large nucleic acid molecules (e.g., cellular genomic DNA) from a nucleic acid sample to obtain polynucleotides in the desired size range. Fragmentation may be random or specific, such as achieved using restriction endonuclease digestion. Random fragmentation methods may include, for example, limited DNase digestion, alkaline treatment, and physical shearing. Fragmentation can also be accomplished by any of a number of methods known to those skilled in the art. For example, fragmentation can be achieved by mechanical means, including but not limited to nebulization, sonication, and water shearing.

In some embodiments, the sample nucleic acid is obtained from unfragmented cfDNA. For example, cfDNA typically exists in fragments of less than about 300 base pairs, and consequently fragmentation is generally not required to generate sequencing libraries using cfDNA samples.

Typically, whether the polynucleotide is forced to fragment (e.g., fragmented in vitro) or exists naturally as fragments, it is converted to blunt-ended DNA with a 5'-phosphate and a 3'-hydroxyl. For example, standard protocols, such as sequencing protocols using the Illumina platform, require users to end-repair sample DNA, purify end-repaired products prior to dA-tailing, and purify dA-tailing products prior to the adapter ligation step of library preparation. Instruct.

In various embodiments, verification of sample integrity and sample tracking can be accomplished by sequencing a mixture of sample genomic nucleic acids, e.g., cfDNA, and accompanying marker nucleic acids introduced into the sample, e.g., prior to processing.

Sequencing techniques

The disclosed sequencing systems and methods are compatible with any sequencing technique based on optical detection, such as next-generation sequencing (NGS), fluorescence in situ sequencing (FISSEQ), and massively parallel signature sequencing (MPSS). In one embodiment, the disclosed systems and methods allow multiple samples to be separated individually into genomic molecules in a single sequencing run (i.e., singleplex sequencing), or as pooled samples containing indexed genomic molecules (e.g., multiplex sequencing). It is compatible with NGS technology that allows for sequencing. These methods can generate up to hundreds of millions of DNA sequence reads.

The disclosed technology is disclosed in U.S. Patent Application Publication No. 2007/0166705, 2006/0188901, 2006/0240439, 2006/0281109, 2005/0100900, U.S. Patent No. 7,057,026, PCT Application Publication No. WO 2005/065814, WO 2006/0 64199, and WO 2007 Sequencing reactions can be implemented such as incorporating the synthetic sequencing methods described in /010251, the disclosure of which is incorporated herein by reference in its entirety. In some embodiments, the sequencer may implement sequencing-by-synthesis methods similar to those used in the HiSeq, MiSeq, or HiScanSQ systems from Illumina (San Diego, CA).

Alternatively, sequencing by ligation techniques can be used as disclosed techniques, such as those described in U.S. Pat. Nos. 6,969,488, 6,172,218 and 6,306,597, the disclosures of which are incorporated herein by reference in their entirety. Sequencing by ligation techniques uses DNA ligase to incorporate oligonucleotides and confirm the incorporation of these oligonucleotides.

The disclosed technology includes the sequencing-by-hybridization platform of Affymetrix Inc. (Sunnyvale, CA) and the sequencing-by-synthesis platform of 454 Life Sciences (Bradford, CT) and Helicos Biosciences (Cambridge, MA), Applied Biosystems (Foster City, CA) )'s sequencing platform by ligation, or with some commercially available sequencing techniques, such as Pacific Biosciences' SMRT technology.

In one exemplary, but non-limiting, embodiment, the methods described herein include Illumina's synthetic sequencing and reversible terminator-based sequencing chemistry (e.g., as described in Bentley et al., Nature 6:53-59 [2009]). It includes obtaining sequence information about nucleic acids using (such as). Illumina's sequencing technology can involve attaching fragmented genomic DNA to a flat, optically clear surface bound to oligonucleotide anchors. For example, template DNA is end repaired to generate a 5'-phosphorylated blunt end, and the polymerase activity of the Klenow fragment is used to add a single A base to the 3' end of the blunt phosphorylated DNA fragment. This addition prepares a DNA fragment for ligation to an oligonucleotide adapter with an overhang of a single T base at the 3' end to increase ligation efficiency. The adapter oligonucleotide is complementary to the flowcell anchor oligo. Under limiting dilution conditions, single-stranded template DNA modified with adapters is added to the flowcell and immobilized on anchor oligos through hybridization. The attached DNA fragments are extended and bridge-amplified to create an ultra-high-density sequencing flowcell with hundreds of millions of clusters, each containing about 1,000 copies of the same template. In one embodiment, randomly fragmented genomic DNA is amplified using PCR prior to being subjected to cluster amplification. Alternatively, amplification-free (e.g., no PCR) genomic library preparation is used, and randomly fragmented genomic DNA is enriched using cluster amplification only (Kozarewa et al., Nature Methods 6:291-295 [2009] ). Sequencing reactions by synthesis can use reversible terminators with removable fluorescent dyes. Short sequence reads of approximately tens to hundreds of base pairs are aligned to a reference genome and the unique mapping of the short sequence reads to the reference genome is identified. After the first read is complete, the template can be regenerated in situ to activate a second read at the opposite end of the fragment. Therefore, single-end or double-end sequencing of DNA fragments can be used. More information about paired-end sequencing can be found in U.S. Patent No. 7601499 and U.S. Patent Publication No. 2012/0,053,063, which are incorporated by reference.

In some embodiments, the sequencing platform by synthesis by Illumina includes clustering of fragments. Clustering is a process in which each fragment molecule is amplified isothermally. In some embodiments, a fragment has two different adapters attached to two ends of the fragment, the adapters allowing the fragment to hybridize to two different oligos on the surface of the flowcell lane. The fragment further comprises or is joined to two index sequences at the two ends of the fragment, where the index sequences provide labels to identify different samples in multiplex sequencing.

In some implementations, the flow cell for clustering on the Illumina platform is a glass slide with lanes. Each lane is a glass channel coated with two types of oligomers. Hybridization is activated by the first of two types of oligos present on the surface. This oligo is complementary to the first adapter at one end of the fragment. The polymerase produces complementary strands of the hybridized fragment. The double-stranded molecule is denatured and the original template strand is washed away. The remaining strand is clonally amplified through bridge application in parallel with many other remaining strands.

In bridge amplification, the strand is folded and a second adapter region at the second end of the strand hybridizes with a second type of oligo on the flow cell surface. Polymerase produces complementary strands to form double-stranded cross-linked molecules. This double-stranded molecule is denatured to produce two single-stranded molecules that are linked to the flow cell via two different oligos. This process is then repeated continuously and occurs simultaneously for millions of clusters, resulting in clonal amplification of all fragments. After bridge amplification, the reverse strand is cleaved and washed, leaving only the forward strand. The 3' end is blocked to prevent unwanted priming.

Sequencing begins by extending the first sequencing primer after clustering to generate the first reads. In each cycle, fluorescently tagged nucleotides compete to be added to the growing chain. Depending on the sequence of the template, only one is incorporated. After adding each nucleotide, the cluster is excited by a light source and a characteristic fluorescence signal is emitted. The read length is determined by the number of cycles. Base calling is determined based on the emission wavelength and signal intensity. For a given cluster, all identical strands are read simultaneously. Hundreds of millions of clusters, or tens to hundreds of millions of clusters, are sequenced in massively parallel fashion. Once the first read is complete, the read product is washed away.

In a process involving two index primers, the index 1 primer is introduced and hybridized to the index 1 region of the template. The index region provides identification of fragments, which is useful for demultiplexing samples during multiplex sequencing. Index 1 reads are generated similarly to the first read. After the index 1 read is complete, the read product is washed away and the 3' end of the strand is deprotected. The template strand is then folded and bound to the second oligo on the flow cell. Index 2 sequences are read in the same way as index 1. The index 2 read product is then washed away upon completion of the step.

After the two index reads, read 2 begins by extending the second flowcell oligo using polymerase to form a double-stranded bridge. This double-stranded DNA is denatured and the 3' end is blocked. The original front strand is cut and washed away, leaving the opposite strand. Read 2 begins with introduction of the Read 2 sequencing primer. As with read 1, the sequencing step is repeated until the desired length is reached. The product of read 2 is washed away. This entire process generates millions of reads representing every fragment. Sequences in the pooled sample library are separated based on unique indices introduced during sample preparation. For each sample, reads with similar base call ranges are clustered locally. Forward and reverse reads are paired to produce contiguous sequences. These contiguous sequences are aligned to a reference genome for variant identification.

computing system

In some embodiments, the disclosed systems and methods may include approaches for moving or distributing certain sequence data analysis features and sequence data repositories to a cloud computing environment or cloud-based network. User interaction with sequencing data, genomic data, or other types of biological data may be mediated through a central hub that stores and controls access to various interactions with the data. In some embodiments, a cloud computing environment may also provide for sharing of protocols, analysis methods, libraries, sequence data, as well as distributed processing for sequencing, analysis, and reporting. In some embodiments, a cloud computing environment facilitates modification or annotation of sequence data by users. In some embodiments, the systems and methods can be implemented in a computer browser, on demand or online.

In some embodiments, software written to perform the methods described herein includes computer readable memory, such as a memory, CD-ROM, DVD-ROM, memory stick, flash drive, hard drive, SSD hard drive, server, mainframe storage system, etc. Available to be stored in some form of media.

In some embodiments, methods may be written in any of a variety of suitable programming languages, such as compiled languages such as C, C#, C++, Fortran, and Java. Other programming languages may be scripting languages such as Perl, MatLab, SAS, SPSS, Python, Ruby, Pascal, Delphi, R, and PHP. In some embodiments, the methods are written in C, C#, C++, Fortran, Java, Perl, R, Java, or Python. In some embodiments, the method may be a stand-alone application with data entry and data display modules. Alternatively, the method may be a computer software product and the distributed object may include a class containing an application that includes the computational methods described herein.

In some embodiments, the methods can be integrated into existing data analysis software, such as that found on sequencing instruments. Software, including the computer-implemented methods described herein, may be installed directly on a computer system or stored indirectly on a computer-readable medium and loaded into the computer system as needed. Additionally, these methods may be located on computers remote from where the data is generated, such as software on servers maintained in a different location relative to where the data is generated, such as provided by a third-party service provider.

A black device, desktop computer, laptop computer, or server that may contain a processor operably in communication with accessible memory containing instructions for implementation of the systems and methods. In some embodiments, a desktop computer or laptop computer is in operative communication with one or more computer-readable storage media or devices and/or output devices. Black devices, desktop computers, and laptop computers can operate with a variety of computer-based operating languages, such as those used on Apple-based computer systems or PC-based computer systems. The assay instrument, desktop and/or laptop computer, and/or server system may further provide a computer interface for creating or modifying experimental definitions and/or conditions, viewing data results, and monitoring experimental progress. In some embodiments, the output device is a graphical user interface such as a computer monitor or computer screen, a printer, a portable device such as a personal digital assistant (e.g., PDA, Blackberry, iPhone), a tablet computer (e.g., an iPAD), a hard drive, , servers, memory sticks, flash drives, etc.

A computer-readable storage device or medium can be any device such as a server, mainframe, supercomputer, magnetic tape system, etc. In some embodiments, the storage device may be located on-site in a location close to the calibration device, such as adjacent to or in close proximity to the calibration device. For example, the storage device may be located in the same room, in the same building, in an adjacent building, on the same floor of the building, on a different floor of the building, etc., relative to the calibration device. In some embodiments, the storage device may be located external to or remote from the assay device. For example, the storage device may be located in a different part of the city, a different city, a different state, a different country, etc., relative to the test device. In embodiments where the storage device is located remotely from the calibration device, communication between the calibration device and one or more of a desktop, laptop, or server typically occurs via an Internet connection, wirelessly through an access point, or via a network cable. In some embodiments, the storage device may be maintained and managed by an individual or entity directly associated with the assay device, while in other embodiments the storage device may be maintained and managed by a third party, generally at a remote location from the individual or entity associated with the assay device. It can be maintained and managed by the owner. In embodiments, the output device can be any device for visualizing data as described herein.

Use the assay instrument, desktop, laptop and/or server system as such to store and/or store computer-implemented software programs that include computer code for performing and implementing the computational methods described herein, data for use in implementing the computational methods, etc. Or you can search. One or more of the assay devices, desktops, laptops and/or servers are one for storing and/or retrieving software programs including computer code for performing and implementing the computational methods described herein, data for use in implementing the computational methods, etc. It may include one or more computer-readable storage media. Computer-readable storage media may include, but are not limited to, one or more of a hard drive, SSD hard drive, CD-ROM drive, DVD-ROM drive, floppy disk, tape, flash memory stick, or card. Additionally, a network including the Internet may be a computer-readable storage medium. In some embodiments, the computer-readable storage medium includes a computational resource storage accessible by a computer network, for example, over the Internet or a corporate network provided by a service provider, other than a local desktop or laptop computer located remotely from the device. refers to

In some embodiments, a computer-readable storage medium for storing and/or retrieving a computer-implemented software program, including computer code for performing and implementing the computational methods described herein, data for use in implementing the computational methods, etc. is operated and maintained by the Service Provider in operational communication with the device, desktop, laptop and/or server system via an Internet connection or network connection.

In some embodiments, the hardware platform for providing the computational environment includes a processor (i.e., CPU), where processor time and memory layout, such as random access memory (i.e., RAM), are system considerations. For example, small computer systems offer inexpensive, fast processors and large memory and storage capabilities. In some embodiments, a graphics processing unit (GPU) may be used. In some embodiments, a hardware platform for performing the computational methods described herein includes one or more computer systems equipped with one or more processors. In some embodiments, small computers are clustered together to create a supercomputer network.

In some embodiments, the computational methods described herein are performed on a collection of interconnected or interconnected computer systems capable of running various operating systems in a coordinated manner (i.e., grid technology). For example, the CONDOR framework (University of Wisconsin-Madison) and systems available through United Devices are examples of the coordination of multiple stand-alone computer systems for the purpose of processing large amounts of data. These systems can provide a Perl interface for submitting, monitoring, and managing large-scale sequencing jobs on a cluster in a serial or parallel configuration.

Justice

Unless otherwise defined, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this disclosure pertains. See, for example, Singleton et al., Dictionary of Microbiology and Molecular Biology 2nd ed., J. Wiley & Sons (New York, NY 1994); See Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press (Cold Spring Harbor, NY 1989). For the purposes of this disclosure, the following terms are defined below.

As used herein, “nucleotide” includes a nitrogen-containing heterocyclic base, a sugar, and one or more phosphate groups. Nucleotides are the monomeric units of nucleic acid sequences. Examples of nucleotides include, for example, ribonucleotides or deoxyribonucleotides. In ribonucleotides (RNA) the sugar is ribose, and in deoxyribonucleotides (DNA) the sugar is deoxyribose, that is, a sugar without the hydroxyl group present at the 2' position of the ribose. The nitrogen-containing heterocyclic base can be a purine base or a pyrimidine base. Purine bases include adenine (A) and guanine (G), and modified derivatives or analogs thereof. Pyrimidine bases include cytosine (C), thymine (T), and uracil (U) and their modified derivatives or analogs. The C-1 atom of deoxyribose is bonded to N-1 of pyrimidine or N-9 of purine. The phosphate group may be in mono-, di- or tri-phosphate form. These nucleotides may be natural nucleotides, but it should be further understood that non-natural nucleotides, modified nucleotides or analogs of the foregoing nucleotides may also be used.

As used herein, “nucleobase” is a heterocyclic base such as adenine, guanine, cytosine, thymine, uracil, inosine, xanthine, hypoxanthine, or a heterocyclic derivative, analog, or tautomer thereof. Nucleobases can occur naturally or be synthesized. Non-limiting examples of nucleobases include adenine, guanine, thymine, cytosine, uracil, xanthine, hypoxanthine, 8-azapurine, purine substituted at the 8 position with methyl or bromine, 9-oxo-N6-methyladenine, 2-amino. Adenine, 7-deazaxanthin, 7-deazaguanine, 7-deaza-adenine, N4-ethanocytosine, 2,6-diaminopurine, N6-ethano-2,6-diaminopurine, 5- Methylcytosine, 5-(C3-C6)-alkynylcytosine, 5-fluorouracil, 5-bromouracil, thiouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, iso cytosine, isoguanine, inosine, 7,8-dimethylalloxazine, 6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, ethenoadenine, and all incorporated herein by reference in their entirety. , U.S. Patent Nos. 5,432,272 and 6,150,510 and PCT Applications WO 92/002258, WO 93/10820, WO 94/22892, and WO 94/24144, and Fasman ("Practical Handbook of Biochemistry and Molecular Biology", pp.385-394, 1989, CRC Press, Boca Raton, LO).

The term "nucleic acid" or "polynucleotide" refers to a polymer of deoxyribonucleotides or ribonucleotides in single or double stranded form and, unless otherwise limited, known natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides. analogs such as peptide nucleic acids (PNAs) and phosphorothioate DNA. Unless otherwise specified, a particular nucleic acid sequence includes its complementary sequence. Nucleotides include ATP, dATP, CTP, dCTP, GTP, dGTP, UTP, TTP, dUTP, 5-methyl-CTP, 5-methyl-dCTP, ITP, dITP, 2-amino-adenosine-TP, 2-amino-deoxy Adenosine-TP, 2-thiotimidine triphosphate, pyrrolo-pyrimidine triphosphate, and 2-thiocitidine, as well as alpha-thiothiotriphosphate for all of the above bases, and 2'-O for all of the above. -Methyl-ribonucleotide triphosphate. Modified bases include, but are not limited to, 5-Br-UTP, 5-Br-dUTP, 5-F-UTP, 5-F-dUTP, 5-propynyl dCTP, and 5-propynyl-dUTP.

The polymerases used are generally enzymes that combine 3'-OH, 5'-triphosphate nucleotides, oligomers, and their analogs. Polymerases include DNA-dependent DNA polymerase, DNA-dependent RNA polymerase, RNA-dependent DNA polymerase, RNA-dependent RNA polymerase, T7 DNA polymerase, T3 DNA polymerase, T4 DNA polymerase, and T7 RNA polymerase. , T3 RNA polymerase, SP6 RNA polymerase, DNA polymerase I, Klenow fragment, Thermophilus aquaticus DNA polymerase, Tth DNA polymerase, VentR® DNA polymerase (New England Biolabs), Deep VentR® DNA polymerase (New England Biolabs), Bst DNA polymerase large fragment, Stoeffel fragment, 90N DNA polymerase, 90N DNA polymerase, Pfu DNA polymerase, TfI DNA polymerase, Tth DNA polymerase, RepliPHI Phi29 polymerase, TIi DNA polymerase, eukaryotes DNA polymerase beta, telomerase, Therminator™ polymerase (New England Biolabs), KOD HiFi™ DNA polymerase (Novagen), KOD1 DNA polymerase, Q-beta replicase, terminal transferase, AMV reverse transcriptase, M- MLV reverse transcriptase, Phi6 reverse transcriptase, HIV-1 reverse transcriptase, novel polymerases discovered through bioprospecting, and polymerases cited in US 2007/0048748, US 6,329,178, US 6,602,695, and US 6,395,524 (incorporated by reference) Including but not limited to this. These polymerases include wild type, mutant isoforms, and genetically engineered variants. “Encoding” or “parse” is a verb that refers to transferring from one format to another, and refers to transferring the genetic information of a target template sequence to a reporter array.

As used herein, the terms “well,” “cavity,” and “chamber” are used synonymously and refer to individual features defined in a device that can contain a fluid (e.g., liquid, gel, gas). Examples of arrays of the present device may have one or multiple wells. Additionally, it should be understood that the cross-section of the well taken parallel to the surface of the substrate that at least partially defines the well may be curved, square, polygonal, hyperbolic, conical, angled, or the like.

A “light source” can be any device capable of emitting energy along the electromagnetic spectrum. The light source may be visible (VIS), ultraviolet (UV), and/or infrared (IR). “Visible light” (VIS) generally refers to the band of electromagnetic radiation with a wavelength of about 400 nm to about 750 nm. “Ultraviolet (UV) light” generally refers to electromagnetic radiation with wavelengths shorter than visible light, or in the range of about 10 nm to about 400 nm. “Infrared” or infrared radiation (IR) generally refers to electromagnetic radiation having a wavelength greater than the visible range, or from about 750 nm to about 50,000 nm. The light source may provide full spectrum light. The light source can output light at a selected wavelength or range of wavelengths. In some embodiments of the invention, the light source may be configured to provide light above or below a predetermined wavelength or may provide light within a predetermined range. A light source may be used with a filter to selectively transmit or block light of selected wavelengths from the light source. The light source may be connected to a power source by one or more electrical connectors; The light source array may be connected in series or parallel to a power source. The power source may be a battery, a vehicle electrical system, or a building electrical system. The light source may be connected to a power source via control electronics (control circuit); The control electronics may include one or more switches. One or more switches may be automated, controlled by sensors, timers, or other inputs, or controlled by a user or a combination thereof. For example, a user can operate a switch to turn on a UV light source; The light source can be applied continuously until turned off or pulsed (repeated on/off cycles) until turned off. In some embodiments, the light source can be switched from a continuously on state to a pulsed state or vice versa. In some embodiments, a light source can be configured to brighten or dim over time.

For operation, the light source can be connected to a power source capable of providing sufficient power to illuminate the sample. Control electronics may be used to turn the power on or off based on user input or other input, and may also be used to regulate the power to an appropriate level (e.g., to control the brightness of the output light). Control electronics may be configured to turn the light source on and off as desired. The control electronics may include switches for manual, automatic or semi-automatic operation of the light source. The one or more switches may be, for example, transistors, repeaters, or electromechanical switches. In some embodiments, the control circuit may further include an AC-DC and/or DC-DC converter to convert the voltage from the voltage source to an appropriate voltage for the light source. The control circuit may include a DC-DC regulator for voltage regulation. The control circuit may further include a timer and/or other circuit elements for applying a voltage to the optical filter for a fixed period of time after receiving the input. Switches can be activated manually or automatically according to predetermined conditions or timers. For example, control electronics may process information such as user input, stored commands, etc.

One or more of a plurality of light sources may be provided. In some embodiments, each of the plurality of light sources can be the same. Alternatively, one or more of the light sources may be different. The optical characteristics of the light emitted by the light source may be the same or different. The plurality of light sources may or may not be independently controlled. One or more characteristics of the light source may or may not be controlled, including but not limited to whether the light source is turned on or off, the brightness of the light source, the wavelength of the light, the intensity of the light, the angle of illumination, the location of the light source, or a combination thereof. .

In some embodiments, the light output from the light source is from about 350 nm to about 750 nm, or any amount or range in between, for example from about 350 nm to about 360, 370, 380, 390, 400, 410, 420. , 430 or about 450 nm, or any amount or range in between. In other embodiments, the light from the light source is between about 550 and about 700 nm, or any amount or range in between, such as about 550 to about 560, 570, 580, 590, 600, 610, 620, 630, It may be 640, 650, 660, 670, 680, 690 or about 700 nm, or any amount or range in between. In some embodiments, the wavelength of light produced by the light source may vary, for example in the range of 400 nm to 800 nm. In some embodiments, the wavelength of light produced by the light source is about 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570. , 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or It can be a number or a range between two of the values. In some embodiments, the wavelength of light produced by the light source is at least or at most 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560. , 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm It can be. Light sources can emit electromagnetic waves of any spectrum. In some embodiments, the light source can have a wavelength that falls between 10 nm and 100 μm. In some embodiments, the wavelength of light may fall between 100 nm and 5000 nm, 300 nm and 1000 nm, or 400 nm and 800 nm. In some embodiments, the wavelength of light is 10 nm, 100 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 800 nm, 900 nm, 1000 nm, 1100 nm, 1200 nm, 1300 nm. , may be less than and/or equal to 1500 nm, 1750 nm, 2000 nm, 2500 nm, 3000 nm, 4000 nm, or 5000 nm.

In one example, the light source is a light emitting diode (LED) (e.g., gallium arsenide (GaAs) LED, aluminum gallium arsenide (AlGaAs) LED, gallium arsenide phosphide (GaAsP) LED, aluminum gallium indium phosphide (AlGaInP)) LED, gallium ( III) phosphide (GaP) LED, indium gallium nitride (InGaN)/gallium(III) nitride (GaN) LED, or aluminum gallium phosphide (AlGaP) LED). In another example, the light source is a laser, such as a Vertical Cavity Surface Emitting Laser (VCSEL) or Indium-Gallium-Aluminum-Phosphide (InGaAIP) laser, Gallium-Arsenic Phosphide/Gallium Phosphide (GaAsP/GaP) It may be a laser, or another suitable emitter such as a gallium-aluminum-arsenide/gallium-aluminum-arsenide (GaAIAs/GaAs) laser. Other examples of light sources include electrostimulated light sources (e.g., cathode ray light, electrostimulated light (ESL bulbs), cathode ray tubes (CRT monitors), nixie tubes), incandescent light sources (e.g., carbon button lamps, conventional incandescent light bulbs, Halogen lamps, Globar, Nernst lamps), electroluminescent (EL) light sources (e.g. light-emitting diodes - organic light-emitting diodes, polymer light-emitting diodes, solid-state lights, LED lamps, electroluminescent sheets, electroluminescent wires), gas discharge light sources (e.g. For example, fluorescent lamps, induction lamps, hollow cathode lamps, neon and argon lamps, plasma lamps, xenon flash lamps), or high intensity discharge light sources (for example, carbon arc lamps, ceramic discharge metal halide lamps, mercury medium arc iodide lamps) , mercury vapor lamp, metal halide lamp, sodium vapor lamp, xenon arc lamp). Alternatively, the light source may be bioluminescent, chemiluminescent, phosphorescent, or fluorescent.

Optical filters can be tuned in terms of transparency or haze, translucency, transparency or opacity, light transmission (LT), switching speed, durability, photostability, contrast ratio, and light transmission state (e.g., dark or light). “Light Transmission” (LT) refers to the amount of light that transmits or passes through an optical filter, or a device or apparatus containing the same. LT can be expressed in terms of light transmission and/or a change in a particular type of light or wavelength of light (e.g., from about 10% visible light transmittance (LT) to about 90% LT, etc.). LT may alternatively be expressed as absorbance and may include reference to one or more wavelengths that are selectively absorbed. According to some embodiments, the optical filter has less than 80%, or less than 70%, or less than 60%, or less than 50%, or less than 40%, or less than 30%, or less than 20%, or less than 10% in one state. It may be selected or configured to have an LT of less than or an amount or range in between. According to some embodiments, the optical filter has greater than 80%, or greater than 70%, or greater than 60%, or greater than 50%, or greater than 40%, or greater than 30%, or greater than 20%, or greater than 10% in other states. , or an amount or range in between.

The filter may be a bandpass filter and may have a peak transmittance of various wavelengths ranging from 400 nm to 800 nm. In some embodiments, the peak transmittance is 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or between any two of these values. It may be a number or range of or approximately these values. In some embodiments, the peak transmittance is at least or at most 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590 , 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm. The transmission window width of the filter may vary, for example from 1 nm to 50 nm. In some embodiments, the width of the filter is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50 nm, or a number between any two of these values. It can be a range, or it can be approximately these values. In some embodiments, the width of the filter can be at least or at most 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 nm. Short-pass filters can be considered special band-pass filters with a lower limit of the transmission window close to 0 nm. A longpass filter can be considered a special bandpass filter whose upper transmission window is close to infinity. A bandstop filter can be defined as a complement to some bandpass filters.

Nucleosides and nucleotides may be labeled at the sugar or nucleobase sites. The dye may be attached to any position of the nucleotide base, for example via a linker. In certain embodiments, Watson-Crick base pairing can still be performed on the resulting analogs. Specific nucleobase labeling sites include the C5 position of a pyrimidine base or the C7 position of a 7-deaza purine base. Linker groups can be used to covalently attach dyes to nucleosides or nucleotides.

As used herein, the term “covalently attached” or “covalently bonded” refers to the formation of a chemical bond characterized by the sharing of electron pairs between atoms. For example, a covalently attached polymer coating refers to a polymer coating that forms a chemical bond with the functionalized surface of a substrate as compared to attachment to the surface through other means, such as adhesive or electrostatic interactions. It will be appreciated that polymers covalently attached to a surface may be bonded through means other than covalent attachment.

공명 에너지 전달로도 알려짐)을 통해 검출성 신호를 변형시킨다.Nucleotide analogs can be attached to or associated with a photodetectable label via a linker to provide a detectable signal. In some embodiments, the photodetectable label is a fluorescent compound, such as a small molecule fluorescent label. Fluorescent molecules (fluorophores) suitable as fluorescent labels include 1,5 IAEDANS; 1,8-ANS; 4-methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein; 5-carboxyfluorescein (5-FAM); fluorescein amidite (FAM); 5-carboxynaphthofluorescein; tetrachloro-6-carboxyfluorescein (TET); hexachloro-6-carboxyfluorescein (HEX); 2,7-dimethoxy-4,5-dichloro-6-carboxyfluorescein (JOE); VIC®; NED™; tetramethylrhodamine (TMR); 5-carboxytetramethylrhodamine (5-TAMRA); 5-HAT (hydroxytryptamine); 5-hydroxytryptamine (HAT); 5-ROX (carboxy-X-rhodamine); 6-carboxyrhodamine 6G; 6-JOE; Light Cycler® Red 610; Light Cycler® Red 640; Light Cycler® Red 670; Light Cycler® Red 705; 7-amino-4-methylcoumarin; 7-aminoactinomycin D (7-AAD); 7-hydroxy-4-methylcoumarin; 9-amino-6-chloro-2-methoxyacridine; 6-methoxy-N-(4-aminoalkyl)quinolinium bromide hydrochloride (ABQ); acid fuchsin; ACMA (9-amino-6-chloro-2-methoxyacridine); acridine orange; Acridine Red; acridine yellow; acriflavine; acriflavin poylgen SITSA; AFPs-autofluorescent proteins-(Quantum Biotechnologies); Texas Red; Texas Red-X zygote; Thiadicarbocyanin (DiSC3); Thiazine Red R; thiazole orange; thioflavin 5; Thioflavin S; Thioflavin TCN; thiolite; Thiozol Orange; Tinopol CBS (Calcofluor White); TMR; TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITC (tetramethylrhodamine-isothiocyanate); true blue; True Red; ultra light; Uranine B; UBITEX SFC; WW 781; X-rhodamine; X-rhodamine-5-(and-6)-isothiocyanate (5(6)-XRITC); xylene orange; Y66F; Y66H; Y66W; YO-PRO-1; YO-PRO-3; YOYO-1; Interchelating dyes such as YOYO-3, Sybr Green, and thiazole orange; Members of the Alexa Fluor® dye series (from Molecular Probes/Invitrogen) that cover a broad spectrum and match the dominant output wavelengths of common excitation sources, such as Alexa Fluor 350, Alexa Fluor 405, 430, 488, 500, 514, 532, 546, 555, 568, 594, 610, 633, 635, 647, 660, 680, 700, and 750; Members of the broad-spectrum Cy Dye fluorophore series (GE Healthcare) such as Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7; Members of the Oyster® dye fluorophore (Denovo Biolabels) such as Oyster-500, -550, -556, 645, 650, 656; For example, members of the DY-Labels series (Dyomics) with maximum absorption ranging from 418 nm (DY-415) to 844 nm (DY-831), such as DY-415, -495, -505, -547, -548, -549, -550, -554, -555, -556, -560, -590, -610, -615, -630, -631, -632, -633, -634, -635, -636, -647 , -648, -649, -650, -651, -652, -675, -676, -677, -680, -681, -682, -700, -701, -730, -731, -732, - 734, -750, -751, -752, -776, -780, -781, -782, -831, -480XL, -481XL, -485XL, -510XL, -520XL, -521XL; Members of the ATTO series (ATTO-TEC GmbH) of fluorescent labels, such as ATTO 390, 425, 465, 488, 495, 520, 532, 550, 565, 590, 594, 610, 611X, 620, 633, 635, 637, 647, 647N, 655, 680, 700, 725, 740; Members of the CAL Fluor® series or Quasar® series (Biosearch Technologies) of dyes, such as CAL Fluor® Gold 540, CAL Fluor® Orange 560, Quasar® 570, CAL Fluor® Red 590, CAL Fluor® Red 610, CAL Fluor® Red Including, but not limited to, 635, Quasar® 570, and Quasar® 670. In some embodiments, the first photodetectable label interacts with a second photodetectable moiety to achieve, for example, fluorescence resonance energy transfer (“FRET”;

Modifies the detectable signal through (also known as resonance energy transfer).

Fluorescent labels utilized by the systems and methods disclosed herein can have various peak absorption wavelengths, for example, ranging from 400 nm to 800 nm. In some embodiments, the peak absorption wavelength of the fluorescent label is 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or any of these values. It may be a number or range between two values, or may be approximately these values. In some embodiments, the peak absorption wavelength of the fluorescent label is at least or at most 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, may be 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm .

Fluorescent labels can have various peak emission wavelengths, for example ranging from 400 nm to 800 nm. In some embodiments, the peak emission wavelength of the fluorescent label is 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 nm, or any of these values. It may be a number or range between two values, or may be approximately these values. In some embodiments, the peak emission wavelength of the fluorescent label is at least or at most 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, may be 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, or 800 nm .

Fluorescent labels may have varying Stokes shifts, for example ranging from 10 nm to 200 nm. In some embodiments, the stroke travel is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200 nm, or It may be a number or range between any two of these values, or may be approximately these values. In some embodiments, the stoke movement is at least or at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, or It may be 200 nm.

Two or more fluorescent labels may have overlapping emission spectra and crosstalk may occur. In some embodiments, the distance between the peak emission wavelengths of any two fluorescent labels can vary, for example in the range from 10 nm to 200 nm. In some embodiments, the distance between the peak emission wavelengths of any two fluorescent labels is 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, It may be 170, 180, 190, 200 nm, or a number or range between any two of these values, or may be approximately these values. In some embodiments, the distance between the peak emission wavelengths of any two fluorescent labels is at least or at most 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150. , 160, 170, 180, 190, or 200 nm.

Various types of linkers with various lengths and chemical properties can be used. The term “linker” includes any moiety useful for linking one or more molecules or compounds to each other, to other components of the reaction mixture, and/or to the reaction site. For example, a linker can attach a reporter molecule or “label” (e.g., a fluorescent dye) to a reaction component. In certain embodiments, the linker is substituted or unsubstituted alkyl (e.g., 2 to 5 carbon chains), substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cyclo. It is a member selected from alkyl, and substituted or unsubstituted heterocycloalkyl. In one example, the linker moiety optionally contains at least one heteroatom (e.g., at least one functional group such as ether, thioether, amide, sulfonamide, carbonate, carbamate, urea, and thiourea), and at least selected from straight and branched carbon chains, optionally containing one aromatic, heteroaromatic or non-aromatic ring structure (e.g., cycloalkyl, phenyl). In certain embodiments, synuric chloride, mylamin, diaminopropanoic acid, aspartic acid, cysteine, glutamic acid, pyroglutamic acid, S-acetylmercaptosuccinic anhydride, carbobenzoxylysine, histine, lysine, serine, homo Molecules with trifunctional linking capabilities are used, including but not limited to serine, tyrosine, piperidinyl-1,1-amino carboxylic acid, diaminobenzoic acid, etc. In certain specific embodiments, hydrophilic PEG (polyethylene glycol ) linker is used.

In certain embodiments, the linker is derived from a molecule comprising at least two reactive functional groups (e.g., one at each end), which reactive functional groups are capable of reacting with complementary reactive functional groups on the various reactive moieties or are capable of reacting with one or more reactive functional groups. It can be used to immobilize components at reaction sites. As used herein, “reactive functional group” includes olefins, acetylenes, alcohols, phenols, ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters, amides, cyanates, isocyanates, thiocyanates, isothiocyanates, Amine, hydrazine, hydrazone, hydrazide, diazo, diazonium, nitro, nitrile, mercaptan, sulfide, disulfide, sulfoxide, sulfone, sulfonic acid, sulfinic acid, acetal, ketal, anhydride, sulfate, isofenic acid. Nitrile, amidine, imide, imidate, nitrone, hydroxylamine, oxime, hydroxamic acid, thiohydroxamic acid, allene, ortho ester, sulfite, enamine, phosphoamine, urea, pseudourea, semicarbazide , carbodiimide, carbamate, imine, azide, azo compound, azoxy compound, and nitroso compound. Reactive functional groups also include those used to prepare bioconjugates, such as N-hydroxysuccinimide esters, maleimides, etc.

Cleavable linkers include, but are not limited to, electrophilic cleavable linkers, nucleophilic cleavable linkers, photocleavable linkers, cleavable under reducing conditions (e.g. disulfide or azide containing linkers), cleavable under oxidizing conditions, safety catch. It may be cleavable through the use of a linker or cleavable through a removal mechanism. Using a cleavable linker to attach the dye compound to the substrate moiety allows the label to be removed after detection if necessary, avoiding any interfering signals in downstream steps.

As used herein, an “optical channel” is a predefined profile of optical frequencies (or equivalently wavelengths). For example, the first optical channel may have a wavelength of 500 nm to 600 nm. To take an image in the first optical channel, use a detector that responds only to light between 500 nm and 600 nm, or use a bandpass filter with a transmission window of 500 nm to 600 nm to divide the incoming light into light between 300 nm and 800 nm. Filtering is possible with a responsive detector. The second optical channel may have a wavelength of 300 nm to 450 nm and 850 nm to 900 nm. To take images in a second optical channel, one can use a detector that responds to 300 nm to 450 nm light and another detector that responds to 850 nm to 900 nm light, and then add the detected signals from both detectors. Alternatively, a bandstop filter that rejects 451 nm to 849 nm light can be used in front of a detector that responds to 300 nm to 900 nm light to image in the second optical channel.

Additional notes

The embodiments described herein are exemplary. Modifications, rearrangements, substitutions, etc. can be made to these embodiments and still remain within the teachings described herein. One or more of the steps, processes, or methods described herein may be performed by one or more appropriately programmed processing and/or digital devices.

The various exemplary imaging or data processing techniques described in connection with the embodiments disclosed herein may be implemented in electronic hardware, computer software, or a combination of the two. To illustrate this interchangeability of hardware and software, various illustrative components, blocks, modules and steps have been described generally in terms of functionality. Whether such functionality is implemented in hardware or software depends on the specific application and design constraints imposed on the overall system. The described functionality may be implemented in varying ways for each particular application, but such implementation decisions should not be construed as causing a departure from the scope of the present disclosure.

Various example detection systems described in connection with embodiments disclosed herein may be implemented using a processor, digital signal processor (DSP), application specific integrated circuit (ASIC), field programmable gate array (FPGA), or other programmable logic device configured with specific instructions. It may be implemented or performed by a machine such as a separate gate or transistor logic, a separate hardware component, or any combination thereof designed to perform the functions described herein. The processor may be a microprocessor, but alternatively the processor may be a controller, microcontroller, or state machine, a combination thereof, etc. The processor may also be implemented as a combination of computing devices, such as a combination of a DSP and a microprocessor, a plurality of microprocessors, one or more microprocessors coupled with a DSP core, or any other such configuration. For example, the systems described herein may be implemented using individual memory chips, portions of the memory of a microprocessor, flash, EPROM, or other types of memory.

Elements of the methods, processes, or algorithms described in connection with the embodiments disclosed herein may be implemented directly in hardware, software modules executed by a processor, or a combination of the two. Software modules may reside in RAM memory, flash memory, ROM memory, EPROM memory, EEPROM memory, registers, hard disk, removable disk, CD-ROM, or any other form of computer-readable storage medium known in the art. An example storage medium can be coupled to the processor such that the processor can read information from the storage medium and write information to the storage medium. Alternatively, the storage medium may be integrated into the processor. The processor and storage media may reside in an ASIC. A software module may include computer-executable instructions that cause a hardware processor to execute computer-executable instructions.

Conditional language used herein, such as “can,” “might,” “may,” “for example,” etc., is used unless otherwise specifically stated. Unless otherwise understood within the context in which it is used, it is generally intended to convey that certain embodiments include certain features, elements and/or states but not other embodiments. Accordingly, such conditional language generally requires that a feature, element, and/or state be in some way required in one or more embodiments, or that one or more embodiments require that such feature, element, and/or state be included or performed in a particular embodiment. It is not implied that it necessarily contains logic for determining whether or not author input or prompts are present. The terms “comprising,” “including,” “having,” “involving,” etc. are synonymous and are used in an open-ended and inclusive manner to refer to additional elements, features, acts, operations, etc. Don't rule it out. Additionally, the term "or" is used in an inclusive sense (rather than an exclusive sense), so that, for example, when used to concatenate a list of elements, the term "or" means one, some, or all of the elements in the list. do.

A conjunction, such as the phrase “at least one of It is understood as a context commonly used to indicate that it can be or Z). Accordingly, such conjunctions are generally not intended to and should not imply that a particular embodiment requires each of at least one of X, at least one of Y, or at least one of Z to be present.

Terms such as "about" or "approximately" are synonyms and are used to indicate that the value modified by the term has an understood range associated therewith, wherein the range is ±20%, ±15%, ±10%, It may be ±5%, or ±1%. The term "substantially" is used to indicate that a result (e.g., a measured value) is close to a target value, where close means, for example, that the result is within 80% of the value, within 90% of the value, or close to the target value. It may mean within 95% of the value, or within 99% of the value.

Unless explicitly stated otherwise, articles such as “a” or “an” should generally be interpreted as including one or more of the described items. Accordingly, phrases such as “a device consisting of” or “a device for” are intended to include one or more of the recited devices. Such one or more requoting devices may be collectively configured to perform the specified requoting. For example, a “processor performing recitation A, B, and C” may include a first processor configured to perform recitation A operating in conjunction with a second processor configured to perform recitation B and C.

Although the above detailed description illustrates, describes, and points out novel features applicable to example embodiments, various omissions, substitutions, and changes in the form and details of the illustrated devices or algorithms may be made without departing from the spirit of the present disclosure. It will be understood that there is. As will be appreciated, certain embodiments described herein may be implemented in forms that do not provide all of the features and advantages described herein because some features may be used or practiced separately from other features. All changes that come within the meaning and equivalency of the claims are included within their scope.

It should be understood that all combinations of the foregoing concepts (unless such concepts are mutually contradictory) are considered part of the inventive subject matter disclosed herein. In particular, any combination of claims appearing at the end of this disclosure is considered to be part of the subject matter disclosed herein.

Claims (30)

A system for identifying nucleotides in a nucleic acid sequence bound to a substrate, comprising:
a first detector configured to detect light in a first wavelength range;
a second detector configured to detect light in a second wavelength range;
A light source including a laser or light-emitting diode that outputs light at an optical frequency; and
producing light at optical frequencies to stimulate emission from nucleic acid sequences on the substrate;
a processor configured to identify nucleotides in a nucleic acid sequence based on whether the emission is received at a first detector, a second detector, both the first and second detectors, or neither the first detector nor the second detector; , system. According to paragraph 1,
a first nucleotide coupled to a first fluorescent label;
a second nucleotide coupled to a second fluorescent label;
a third nucleotide coupled to a third fluorescent label; and
It further comprises a fourth nucleotide that is not coupled to the fluorescent label,
Here the light source is
exciting the first fluorescent label to emit light that can be detected by a first detector;
exciting the second fluorescent label to emit light that can be detected by a second detector;
A system configured to excite a third fluorescent label to emit light that can be detected by both the first and second detectors. 3. The system of claim 2, wherein the fluorescent label is selected from the group consisting of polymethine derivatives, coumarin derivatives, benzopyran derivatives, and chromenoquinoline derivatives. The system of claim 2, wherein the fluorescent label is selected from the group consisting of:

, and . 3. The system of claim 2, further comprising an additional first nucleotide that is not coupled to a fluorescent label. 3. The system of claim 2, further comprising an additional first nucleotide coupled to an alternative fluorescent label, wherein the alternative fluorescent label cannot be excited by a light source to emit light detectable by the first detector. 3. The method of claim 2, further comprising an additional first nucleotide coupled to the alternative fluorescent label, wherein the alternative fluorescent label is capable of being excited by a light source to emit light detectable by the first detector, and wherein the alternative fluorescent label is capable of emitting light detectable by the first detector. A system wherein the label emits a darker light compared to the first fluorescent label. The method of claim 6, wherein the alternative fluorescent label is In,system. 7. The system of claim 6, wherein the alternate fluorescent label and the first fluorescent label have different fluorescence emission spectra. 3. The method of claim 2, wherein the first fluorescent label has a Stokes shift between 20 nm and 50 nm, the second fluorescent label has a Stokes shift between 100 nm and 130 nm, and the third fluorescent label has a Stokes shift between 60 nm and 90 nm. system, having a Stokes shift between. 3. The system of claim 2, wherein the first fluorescent label cannot be detected by the second detector and the second fluorescent label cannot be detected by the first detector. 3. The system of claim 2, wherein the first fluorescent label can also be detected by the second detector, or the second fluorescent label can also be detected by the first detector. The system of claim 1, wherein the first wavelength range and the second wavelength range do not overlap. The system of claim 1, wherein the optical frequency corresponds to a wavelength in a predefined range of optical wavelengths, wherein the predefined range includes at least one wavelength shorter than all wavelengths in the first range and the second range. 15. The system of claim 14, wherein the predefined range includes 405 nm to 460 nm. The system of claim 1, wherein the optical frequency corresponds to a wavelength of a predefined range of optical wavelengths, wherein the predefined range includes at least one wavelength that is longer than some wavelength of the first range or the second range. 3. The system of claim 2, wherein the light source is configured to excite the fluorescent label by a two-photon absorption process. The system of claim 1 , wherein the detector comprises a complementary metal-oxide-semiconductor image sensor, a charge coupled device image sensor, a photomultiplier tube, a photodiode, or any combination thereof. The system of claim 1 , further comprising one or more optical filter materials, one or more diffraction gratings, one or more light dispersing elements, or any combination thereof. 3. The system of claim 2, further comprising a polymerase configured to replicate or transcribe a portion of a nucleic acid sequence by incorporating nucleotides. The system of claim 1 , wherein the substrate comprises a plurality of chemically functionalized regions, a plurality of cavities, a plurality of optical resonators, a plurality of optical waveguides, or any combination thereof. 3. The system of claim 2, wherein the nucleotide and fluorescent label are coupled by a cleavable linker. 3. The system of claim 2, wherein the nucleotides are selected from the group consisting of dGTP analogs, dTTP analogs, dUTP analogs, dCTP analogs, and dATP analogs. The method of claim 2, wherein the first nucleotide is a first reversibly blocked nucleotide triphosphate (rbNTP), the second nucleotide is a second rbNTP, the third nucleotide is a third rbNTP, and the fourth nucleotide is a fourth rbNTP. In,system. 25. The system of claim 24, wherein the four rbNTPs are selected from the group consisting of rbATP, rbTTP, rbUTP, rbCTP, and rbGTP. 25. The system of claim 24, wherein each of the four rbNTPs comprises a modified base and a reversible terminator 3' blocker. A method for determining the sequence of a polynucleotide, comprising:
Emitting light at an optical frequency from a light source to the polynucleotide;
determining whether the polynucleotide has a bound fluorescent label that fluoresces under a first wavelength of light, a second wavelength of light, both the first and second wavelengths of light, or is devoid of fluorescence; and
A method comprising identifying a sequence of a polynucleotide based on whether there is detectable emission or no fluorescence in a first wavelength of light, a second wavelength of light, both the first and second wavelengths of light. 28. The method of claim 27, wherein determining whether the polynucleotide has a bound fluorescent label comprises:
determining whether the first nucleotide is coupled to the first fluorescent label;
determining whether a second nucleotide is coupled to a second fluorescent label;
determining whether a third nucleotide is coupled to a third fluorescent label; and
Determining whether the fourth nucleotide is not coupled to the fluorescent label. 28. The method of claim 27, wherein the bound fluorescent label is selected from the group consisting of polymethine derivatives, coumarin derivatives, benzopyran derivatives, and chromenoquinoline derivatives. 28. The method of claim 27, wherein the first fluorescent label has a Stokes shift between 20 nm and 50 nm, the second fluorescent label has a Stokes shift between 100 nm and 130 nm, and the third fluorescent label has a Stokes shift between 60 nm and 90 nm. Having a Stokes shift between, methods.

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